Robust sequential biophysical fractionation of blood plasma to study variations in the biomolecular landscape of systemically circulating extracellular vesicles across clinical conditions

Vergauwen, Glenn; Tulkens, Joeri; Pinheiro, Cláudio; Cobos, Francisco Avila; Dedeyne, Sándor; Scheerder, Marie‐Angélique De; Vandekerckhove, Linos; Impens, Francis; Miinalainen, Ilkka; Braems, Geert; Gevaert, Kris; Mestdagh, Pieter; Vandesompele, Jo; Denys, Hannelore; Wever, Olivier De; Hendrix, An

doi:10.1002/jev2.12122

Cited by 42 publications

(54 citation statements)

References 48 publications

(85 reference statements)

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“…This is in agreement with recent studies showing co-isolation between EVs and LPPs in fresh and processed blood plasma samples [8,9,14]. It has been demonstrated that the combination of different EV isolation methods can strongly reduce the amount of LPPs and can be applied to obtain EVs from blood plasma with high purity, however such procedures may also result in the selection of certain subpopulations of EVs [10,[15][16][17][18].…”

Section: Introductionsupporting

confidence: 91%

“…Circulating EVs are often analyzed by FC and recent studies indicated confounding effects of LPPs present in EV preparations in flow cytometric EV analysis [6][7][8]. Based on the partially overlapping properties of EVs and LPPs, optimized sequential biophysical fractionation protocols were developed to separate LPPs from EVs [10,15,17]. However, in recent studies it has been indicated that also complexes of EV-LPP can be detected in EV samples [8,9,40].…”

Section: Discussionmentioning

confidence: 99%

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Physical association of low density lipoprotein particles and extracellular vesicles unveiled by single particle analysis

Lozano‐Andrés

Enciso-Martinez

Gijsbers

et al. 2022

Preprint

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Extracellular vesicles (EVs) in blood plasma are recognized as potential biomarkers for disease. Although blood plasma is easily obtainable, analysis of EVs at the single particle level is still challenging due to the biological complexity of this body fluid. Besides EVs, plasma contains different types of lipoproteins particles (LPPs), that outnumber EVs by orders of magnitude and which partially overlap in biophysical properties such as size, density and molecular makeup. Consequently, during EV isolation LPPs are often co-isolated. Furthermore, physical EV-LPP complexes have been observed in purified EV preparations. Since co-isolation or association of LPPs can impact single EV-based analysis and biomarker profiling, we investigated whether under physiological conditions LPPs and EVs can associate by using cryo-electron tomography, label-free synchronous Rayleigh and Raman scattering analysis of optically trapped particles and fluorescence-based high resolution single particle flow cytometric analysis. Furthermore, we evaluated the impact on flow cytometric analysis in the absence or presence of different types of LPPs using in vitro spike-in experiments of purified tumor cell line-derived EVs in different classes of purified human LPPs. Based on orthogonal single-particle analysis techniques we demonstrated that EV-LPP complexes can form under physiological conditions. Furthermore, we show that in fluorescence-based flow cytometric EV analysis staining of LPPs, as well as EV-LPP associations can influence EV analysis in a quantitative and qualitative manner. Our findings demonstrate that the biological colloidal matrix of the biofluid in which EVs reside impacts their buoyant density, size and/or refractive index (RI), which may have consequences for down-stream EV analysis.

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Section: Introductionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Physical association of low density lipoprotein particles and extracellular vesicles unveiled by single particle analysis

Lozano‐Andrés

Enciso-Martinez

Gijsbers

et al. 2022

Preprint

Self Cite

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“…However, they did not characterise their EVs, which limits the conclusions we can draw from this. Vergauwen et al [ 36 ] used a combination of SEC and density gradient to isolate EVs from blood plasma obtained from a broad range of patients with ovarian cancer, breast cancer, and HIV. This was in an attempt to define clinical context-dependent variations in the EV RNA profile in cancer patients.…”

Section: Resultsmentioning

confidence: 99%

“…For instance, osteoblastogenesis revealed that BMSCs alter their cargo content in EVs from full-length tRNAs to tRNA-halves as their differentiation progresses [ 62 ]. We have noted the evidence that there are time-dependent changes in the EV tRNA profile of cancer patients and may perhaps reflect clinical changes such as disease burden [ 36 ]. In addition, tRFs have even been shown to possess potential diagnostic potential in a multitude of diseases.…”

Section: Discussionmentioning

confidence: 99%

The Clinical Significance of Transfer RNAs Present in Extracellular Vesicles

Liu

Yang

Asim

et al. 2022

IJMS

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Extracellular vesicles (EVs) are important for intercellular signalling in multi-cellular organisms. However, the role of mature transfer RNAs (tRNAs) and tRNA fragments in EVs has yet to be characterised. This systematic review aimed to identify up-to-date literature on tRNAs present within human EVs and explores their potential clinical significance in health and disease. A comprehensive and systematic literature search was performed, and the study was conducted in accordance with PRISMA guidelines. Electronic databases MEDLINE and EMBASE were searched up until 1 January 2022. From 685 papers, 60 studies were identified for analysis. The majority of papers reviewed focussed on the role of EV tRNAs in cancers (31.7%), with numerous other conditions represented. Blood and cell lines were the most common EV sources, representing 85.9% of protocols used. EV isolation methods included most known methods, precipitation being the most common (49.3%). The proportion of EV tRNAs was highly variable, ranging between 0.04% to >95% depending on tissue source. EV tRNAs are present in a multitude of sources and show promise as disease markers in breast cancer, gastrointestinal cancers, and other diseases. EV tRNA research is an emerging field, with increasing numbers of papers highlighting novel methodologies for tRNA and tRNA fragment discovery.

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“…ultracentrifugation vs. cushion ultracentrifugation) provided no benefit in sample purity. Sequential purification steps have better results when using different physical-chemical properties, such as SEC followed by DGUC, but leads to a low EV recovery (∼1%) (Vergauwen et al , 2021). Polymer-based precipitation (PP) had large amounts of lipoprotein contamination, probably due to their mechanism of enriching for EVs by binding to lipids.…”

Section: Discussionmentioning

confidence: 99%

Meta-analysis refinement of plasma extracellular vesicle composition identifies proplatelet basic protein as a signaling messenger in type 1 diabetes

Vallejo

Sarkar

Elliott

et al. 2022

Preprint

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Extracellular vesicles (EVs) play important roles in cell-to-cell communication and are potential biomarkers as they carry markers of their derived tissues and disease signatures. However, obtaining pure EV preparations from biofluids is challenging due to contaminants with similar physicochemical properties. Here, we performed a meta-analysis of plasma EV proteomics data deposited in public repositories to refine the protein composition of EVs and investigate potential roles in type 1 diabetes development. With the concept that each purification method yields different proportions of distinct contaminants, we grouped proteins into clusters based on their abundance profiles. This allowed us to separate clusters with classical EV markers, such as CD9, CD40, C63 and CD81, from clusters of well-known contaminants, such as serum albumin, apolipoproteins and components of the complement and coagulation pathways. Two clusters containing a total of 1720 proteins combined were enriched with EV markers and depleted in common contaminants; therefore, they were considered to contain bona fide EV components. As possible origins of plasma EVs, these clusters had markers of tissues such as spleen, liver, brain, lungs, pancreas, and blood/immune cells. These clusters were also enriched in cell surface markers CD antigens, and proteins from cell-to-cell communication and signaling pathways, such as chemokine signaling and antigen presentation. We also show that the EV component and type 1 diabetes biomarker, platelet basic protein (PPBP/CXCL7) regulates apoptosis in both beta and macrophage cell lines. Overall, our meta-analysis refined the composition of plasma EVs, reinforcing a primary function as messengers for cell-to-cell communication and signaling. Furthermore, this analysis identifies optimal avenues to target EVs for development of disease biomarkers.

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Robust sequential biophysical fractionation of blood plasma to study variations in the biomolecular landscape of systemically circulating extracellular vesicles across clinical conditions

Cited by 42 publications

References 48 publications

Physical association of low density lipoprotein particles and extracellular vesicles unveiled by single particle analysis

Physical association of low density lipoprotein particles and extracellular vesicles unveiled by single particle analysis

The Clinical Significance of Transfer RNAs Present in Extracellular Vesicles

Meta-analysis refinement of plasma extracellular vesicle composition identifies proplatelet basic protein as a signaling messenger in type 1 diabetes

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